Antigen

Antigen Class

TFs and others

Antigen

Nfia

Cell type

Cell type Class

Neural

Cell type

Retina

MeSH Description

The ten-layered nervous tissue membrane of the eye. It is continuous with the OPTIC NERVE and receives images of external objects and transmits visual impulses to the brain. Its outer surface is in contact with the CHOROID and the inner surface with the VITREOUS BODY. The outer-most layer is pigmented, whereas the inner nine layers are transparent.

Sample

source_name

retina

age

P2

tissue

retina

genotype

Nfia/b/x cKO

chip antibody

NFI (NFIA, NFIB, NFIX)

replicate

1

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Chromatin immunoprecipitation was then performed on the sheared DNA using the iDeal ChIP-seq kit for transcription factors (Diagenode Cat# C01010170). One percent of chromatin was kept aside to be used as an input control. Antibodies against targets used for chromatin immunoprecipitation are NFI (NFIA, NFIB, NFIX), and IgG. Briefly, equal volume of sheared chromatin was incubated overnight with 3 μg of antibody in iC1b buffer with protease inhibitors and BSA and washed DiaMag Protein A-coated magnetic beads (Diagenode Cat# C03010020-220) on a tube rotator at 4°C overnight. The magnetic beads were then washed sequentially with wash buffers iW1, iW2, iW3 and iW4. DNA was then de-crosslinked and eluted for 4 hours at 65C before being purified using IPure beads (Diagenode Cat# C03010014). The purified DNA was then subjected to sequencing library preparation or qPCR analysis. Libraries were prepared from 5ng of DNA using the Ovation Ultralow System V2 (Tecan Genomics Cat# 0344NB-32) and sequenced on the Illumina NextSeq500. Dissociated cells were resuspended in ice-cold PBS containing 0.04% bovine serum albumin (BSA). Cell count and viability were assessed by Trypan blue staining.Trypan blue staining. scATAC-seq was performed using the 10x Genomic single cell ATAC reagent v1.1 kit following the manufacturer's instruction. Briefly, dissociated cells were centrifuged at 300xg for 5 min at 4oC. Cell pellet was resuspended in 100 μl of Lysis buffer, mixed 10x by pipetting and incubated on ice for 3 min. Wash buffer (1 ml) was added to the lysed cells, and cell nuclei were centrifuged at 500xg for 5 min at 40C. Nuclei pellet was re-suspended in 250 μl of 1x Nuclei buffer. Cell nuclei were then counted using Trypan blue. Re-suspended cell nuclei (10-15k) were used for transposition and loaded into the 10x Genomics Chromium Single Cell system. Libraries were amplified with 10 PCR cycles and were sequenced on Illumina NextSeq or NovaSeq with ~200 million reads per library. Sequencing data was processed through the Cell Ranger ATAC 1.1.0 pipeline (10x Genomics) using default parameters. single cell ATACseq

Sequencing Platform

instrument_model

NextSeq 500

mm10

Number of total reads

69796058

Reads aligned (%)

97.0

Duplicates removed (%)

16.9

Number of peaks

10199 (qval < 1E-05)

mm9

Number of total reads

69796058

Reads aligned (%)

96.8

Duplicates removed (%)

17.2

Number of peaks

10237 (qval < 1E-05)